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In this experiment the objective was to determine the optimal temperatures for fungal and human amylase. Four sets of tubes were used, human, fungal, starch with human, and starch with fungal amylase were used. Four water baths were also used with different temperatures at 0°, 40°, 60°, and 95° Celsius. With intervals of two minutes two drops were added to spot plates to their corresponding temperature and amylase. Each place had two drops of iodine as well to observe starch catalysis and measured visually using the Iodine test. The effect of temperature and the ability of amylase to break down starch to maltose was observed. This test turns from yellow to black when there is starch present. The results obtained show the optimal temperature and how each temperature affected amylase activity.

Introduction

Amylase is an enzyme. Enzymes are proteins which can catalyze a reaction. They increase the rate of the reaction, but are not consumed in the reaction (Mitchel, 801). All biological processes including growth, reproduction and metabolism, require a constant supply of energy. The production of this energy is accomplished through the thousands of chemical reactions that occur in cells and is regulated by enzymes (Simms, 45). The use of enzymes in our daily lives has significantly improved our standard of living. For instance, proteases and amylases, which are generated by the body to break down proteins and starches, respectively, are also used commercially to make bread, biscuits and crackers (Simms, 45). Therefore it is important to have a good understanding for future references. Without enzymes, life would not be possible; food would not be converted to energy and our bodies would not be able to replace old, damaged tissues with new, healthy ones (Simms, 45).

Most enzymes are proteins with three-dimensional shapes that are determined by their amino acid sequences, when a substrate (reactant) molecule binds to the highly specific active site of an enzyme, an enzyme-substrate (ES) complex is formed (Simms, 45). That complex makes a chemical reaction which creates a product. Note that the product is not the whole enzyme, in fact it is created at the active site of the enzyme, and then released; allowing the enzymes to be used again.

There are thousands of different enzyme types, each with a specific set of conditions at which it works best, an optimal condition often reflect the environment(s) of the organism(s) in which it is found (Simms, 45). Temperature affects the rate at which substrate and enzyme molecules collide. At temperatures greater than optimal the active site denatures, decreasing or preventing substrate binding; at the other end of the spectrum, low temperatures decrease the movement of molecules, resulting in less contact between enzymes and substrates (Simms, 47). With all this information were able to experiment with the enzyme amylase to see how temperature affected it, as well as, to get the optimal temperature for fungal and human. Therefore the hypothesis chosen was that for enzyme amylase as temperature increases or decreases breakdown would decrease after optimal temperature being at body temperature. An alternative hypothesis is that as temperature increases or decrease breakdown would increase. Human and Fungus should have had different optimal temperatures since fungus amylase survive at different temperature levels than human.

Materials and Methods

Initial preparation. In this experiment everything was set up so there would be no time wasted during the experiment since samples need to be taken every two minutes. Each group tested the optimal temperature for each amylase type (fungal and human). Two spot plates were given, and placed on top of napkins in which temperatures of (0°, 40°, 60°, 90° Celcius) were written on top and on the side time intervals of (0, 2, 4, 6, 8, 10 min) were written. Four Test tubes were used and each was labeled with a different temperature, the group number, the enzyme source, and added 1mL of amylase. Another four tubes were used and labeled with different temperature, enzyme source, group number and the letter S since these had 5 mL of 1% starch solution.

Procedure. For Human amylase, 0.5 mL of saliva and 0.5 mL of distilled water was added into each test tube. For Fungal amylase, 1 mL of fungal amylase was added to each tube. All four test tubes with starch, and all four with amylase was placed to the temperature they had written on. The 0°C was placed into an ice bath, and the 40°C, 60°C, and 95°C, were place into their respective temperature water bath. The tubes were left for five min in their respective temperatures. And two drops of iodine were added to the first row of wells on the spot plate labeled 0 min. After the five minutes of equilibration process, a few drops of starch solution were transferred from each temperature to the first row of the spot plate that was labeled 0 min corresponding with the amylase source. This was repeated every two minutes until reaching ten minutes to fill out all of the wells.

Results

The class obtained 12 datasets; six for human and six for fungal amylase. Since there was a large amount data, an average of the class for human and fungal was used. Group 2 data was used to compare to the class averages.

Figure 1: Human Amylase, Average of all groups

Figure 2: Human Amylase, Group 2 Results

Figure 3: Fungal Amylase, Average of all groups

Figure 4: Fungal Amylase, Group 2 Results

There are some different results but overall the mean value from the class comes very close to groups 2.

Discussion

The results for the 0 min column on all figures should have been very dark showing starch giving a 1 but it seems that both the class average and Group 2 results were not accurate. This could have been due to the fact that iodine was not shaken before put into the plates.

On the 2 min column better results started showing since temperature was included. One can see on all the graphs that higher temperatures show denature as most groups resulted in a one being the darkest color. Although some groups have high numbers the mean gives a better result being between one and two. As time passed and more minutes were able to be recorded it can be seen how on figure 1 the optimal temperature seemed to be 40°C for human. And for figure 3 the optimal temperature seemed to be 60°C for fungal. Since higher temperatures show very high level of starch hydrolysis showing denature and lower temperatures starch hydrolysis are not as high but they were still high. Starch was used as a positive control.

As figure 1 demonstrates, values are very close to each other. There might have been a lost in accuracy due to human error when transferring liquids with the pipette. As well as the time interval when groups had to get a sample of their tubes, since it was very crowded some people took longer to get the sample because they would have to wait until the person in front of them were done. That difference in time would count towards the minutes the sample was on the water bath. For example time interval of 2 min for some would be 2 min and 30 sec for others.

In conclusion the optimal temperature for human amylase was 40°C. Although on figure 2 it might show 60°C as being an optimal temperature this could be due to human error, samples could have been mixed or test were not accurate. The different time intervals could also come into play. But by looking at the mean from figure 1 which shows the class average, it was determined that 40°C was the optimal temperature as it was the clearest color. As for the fungal amylase 60°C was determined to be the optimal temperature by looking at the average as well. On figure 4 there was a strange drop from 0°C to 40°C, compared to figure 3, group 2 might have made a mistake or there seems to be some kind of human error since it is very different to the results from the class average.

Doing the mean of the results of all the groups helped seen the experiment more clear since small errors would be erased when the average was used. If one group made a mistake, by taking the average that mistake would not be noticeable. And comparing them to a group one can conclude where a group made a mistake as previous stated with figure 4.

Due to the data obtained, the null hypothesis was failed to be rejected since body temperature is 37°C and the optimal temperature was 40°C; when temperature changes from optimal temperature, breakdown decreases by showing higher starch hydrolysis. This can be accepted since Enzyme activity is different by changes in temperature; as temperature increases the speed of chemical reactions increases as temperature increases the speed of motion of molecules. This creates more exchanges among the enzyme and its substrate (Judith, 109).

Since there is not enough evidence of 40°C being better than 37°C seeing as 37°C was not tested the hypothesis could not be rejected. This can also be proved by the optimum working temperature is different from enzyme to enzyme. Some are best around 10°C, but others at 80°C. Enzymes in the human body have an optimal temperature of 40°C although others would have slightly higher optimal, for example pepsin works faster at 60°C. (Thomes, 49). As for optimal fungal temperature the hypothesis can be rejected since there is a lot of difference between 60°C and 40°C being the closest to body temperature.